Compositions and Methods for Treating Rotator Cuff Injuries

ABSTRACT

The present invention provides compositions and methods for attaching tendon to bone. The present invention provides compositions and methods for treating rotator cuff injuries. In one embodiment, a method for treating rotator cuff injuries comprises providing a composition comprising PDGF disposed in a biocompatible matrix and applying the composition to at least one site of tendon reattachment on the humeral head.

RELATED APPLICATION DATA

The present application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 60/817,874 filed Jun. 30, 2006.

FIELD OF THE INVENTION

The present invention relates to compositions and methods useful forattaching tendon to bone, particularly for repairing rotator cuffinjury.

BACKGROUND OF THE INVENTION

Hundreds of thousands of people experience tendon ruptures and tendondetachments from bone annually. Rotator cuff tears are among the mostcommon injuries observed by practitioners of sports medicine.Approximately 400,000 rotator cuff repair surgeries are performed in theUnited States annually.

The rotator cuff is a group of four tendons which converge and surroundthe front, back, and top of the head of the humerus shoulder joint.These tendons are connected individually to short muscles that originatefrom the scapula. The muscles are referred to as the “SITS”muscles-supraspinatus, infraspinatus, teres minor and subscapularis. Themuscles function to provide rotation and elevate the arm and givestability to the shoulder joint. When the muscles contract, they pull onthe rotator cuff tendons, causing the shoulder to rotate upward, inward,or outward. There is a bursal sac between the rotator cuff and acromionthat allows the muscles to glide freely when moving.

Rotator cuff tendons are susceptible to tears, which are a common sourceof shoulder pain. The tendons generally tear off at their insertion(attachment) onto the humeral head. Injuries to the rotator cuff may bepresent as complete evulsions, or L- or U-type partial tears. Pain, lossof motion and weakness may occur when one of the rotator cuff tendonstears. When rotator cuff tendons are injured or damaged, the bursa oftenbecomes inflamed and may be an additional source of pain.

Notwithstanding surgical instrumentation and advanced techniques, theincidence of re-injury following repair of the rotator cuff is high,with some estimates approaching 70%. The failure of rotator cuff repairshas been attributed to the poor healing and reattachment of the musclesthat insert on the humeral head. The normal fibrotic and proliferativeresponse among tendon fibroblasts and mesenchymal stem cells isdiminished within the shoulder. This inadequate healing responsetherefore transfers the burden of tendon reattachment and integrity tothe mechanical strength of the sutures. Over time, the sutures breakdown and tear away from bone and/or tendon, causing re-injury of theshoulder. The problem has been documented in numerous studies employingthe use of animal models. Coleman and colleagues report in The Journalof Bone and Joint Surgery 85:2391-2402 (2003) that the repairedinfraspinatus muscle of the shoulder is capable of producing only 63% ofthe normal contraction force normal at 12 weeks after repair using asheep model of chronic injury.

In view of the problems associated with rotator cuff repairs, it wouldbe desirable to provide compositions and methods operable to improve thehealing response associated with rotator cuff repairs. In particular, itwould be desirable to provide compositions and methods which enhancefibrotic and proliferative responses among tendon fibroblasts andmesenchymal stems cells thereby promoting healing of a torn rotator cuffand tendon reattachment to the humeral head.

SUMMARY

The present invention provides compositions and methods for thetreatment and/or repair of damaged tendons. In some embodiments,compositions and methods of the present invention are useful in theattachment or reattachment of tendons to bone, and may be applied to anytendon reattachment. In some embodiments, compositions and methods ofthe present invention enhance tendon attachment to bone by strengtheningthe tendon and/or bone at the site of tendon attachment to the bone.Moreover, the treatment of tendons encompasses application ofcompositions of the present invention to tendons, including damaged orinjured tendons, such as tendons exhibiting tearing, delamination,and/or any other strain or deformation. Tendons which may be reattachedto bone and/or treated by compositions and methods of the presentinvention include, but are not limited to, tendons of the subscapularis,supraspinatus, infraspinatus, teres minor, rectus femoris, tibialisposterior, and quadraceps femoris, as well as the Achilles Tendon,patellar tendon, abductor and adductor tendons, or other tendons of thehip.

In accordance with some embodiments of the present invention, there areprovided compositions and methods for the treatment of rotator cufftears. The present compositions and methods facilitate the healingresponse to rotator cuff repairs and tendon reattachment to the humeralhead.

In one aspect, a composition provided by the present invention forpromoting tendon reattachment to the humeral head comprises a solutioncomprising platelet derived growth factor (PDGF) and a biocompatiblematrix, wherein the solution is disposed in the biocompatible matrix. Insome embodiments, PDGF is present in the solution in a concentrationranging from about 0.01 mg/ml to about 10 mg/ml, from about 0.05 mg/mlto about 5 mg/ml, or from about 0.1 mg/ml to about 1.0 mg/ml. Theconcentration of PDGF within the solution may be within any of theconcentration ranges stated above.

In embodiments of the present invention, PDGF comprises PDGF homodimersand heterodimers, including PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD,and mixtures and derivatives thereof. In one embodiment, PDGF comprisesPDGF-BB. In another embodiment PDGF comprises a recombinant human (rh)PDGF such as recombinant human PDGF-BB (rhPDGF-BB).

In embodiments of the present invention, PDGF comprises PDGF fragments.In one embodiment rhPDGF-B comprises the following fragments: amino acidsequences 1-31, 1-32, 33-108, 33-109, and/or 1-108 of the entire Bchain. The complete amino acid sequence (1-109) of the B chain of PDGFis provided in FIG. 15 of U.S. Pat. No. 5,516,896. It is to beunderstood that the rhPDGF compositions of the present invention maycomprise a combination of intact rhPDGF-B (1-109) and fragments thereof.Other fragments of PDGF may be employed such as those disclosed in U.S.Pat. No. 5,516,896. In accordance with a preferred embodiment, therhPDGF-BB comprises at least 65% of intact rhPDGF-B (1-109).

A biocompatible matrix, according to some embodiments of the presentinvention, comprises a bone scaffolding material. In some embodiments, abone scaffolding material comprises calcium phosphate. Calciumphosphate, in one embodiment, comprises β-tricalcium phosphate (β-TCP)

In another aspect, the present invention provides a compositioncomprising a PDGF solution disposed in a biocompatible matrix, whereinthe biocompatible matrix comprises a bone scaffolding material and abiocompatible binder. The PDGF solution may have a concentration of PDGFas described above. A bone scaffolding material, in some embodiments,comprises calcium phosphate. In one embodiment, a calcium phosphatecomprises a β-TCP. In one aspect, biocompatible matrices may includecalcium phosphate particles with or without biocompatible binders orbone allograft such as demineralized freeze dried bone allograft (DFDBA)or particulate demineralized bone matrix (DBM). In another aspect,biocompatible matrices may include bone allograft such as DFDBA or DBM.

Moreover, a biocompatible binder, according to some embodiments of thepresent invention, comprises proteins, polysaccharides, nucleic acids,carbohydrates, synthetic polymers, or mixtures thereof. In oneembodiment, a biocompatible binder comprises collagen. In anotherembodiment, a biocompatible binder comprises collagen, such as bovine orhuman collagen.

The present invention additionally provides methods of producingcompositions for the reattachment of tendons to bone, the strengtheningof tendon attachment to bone, and/or the treatment of tendons including,but not limited to, those associated with rotator cuff tears. In oneembodiment, a method for producing a composition comprises providing asolution comprising PDGF, providing a biocompatible matrix, anddisposing the solution in the biocompatible matrix.

The present invention also provides methods for the reattachment oftendons to bone, the strengthening of tendon attachment to bone as wellas methods for the treatment of tendons including damaged or injuredtendons, such as those exhibiting tearing, delamination, or any otherstrain or deformation. In one embodiment, a method for attaching atendon to bone and/or strengthening tendon attachment to bone comprisesproviding a composition comprising a PDGF solution disposed in abiocompatible matrix and applying the composition to at least one siteof tendon reattachment on the bone. In another embodiment, a method fortreating rotator cuff tears comprises providing a composition comprisinga PDGF solution disposed in a biocompatible matrix and applying thecomposition to at least one site of tendon reattachment on the humeralhead. In some embodiments, a method for treating a rotator cuff tearfurther comprises disposing at least one bone anchor in the humeralhead, the at least one bone anchor further comprising a PDGF solutiondisposed in a biocompatible matrix, and coupling at least one detachedtendon to the bone anchor.

In another embodiment, a method of treating a tendon comprises providinga composition comprising a PDGF solution disposed in a biocompatiblematrix and applying the composition to a surface of at least one tendon.In some embodiments, the at least one tendon is an injured or damagedtendon, such as tendon exhibiting tearing, delamination, or any otherdeformation.

In some embodiments, the present invention may be used to repair atendon tear that is not at a bone attachment point. Such a tendon tearmay be sutured together with an overlay material which would releasePDGF from the material. In some embodiments, for example, a tear occursin midsubstance ruptures of the Achilles Tendon. Overlay materials fortreating and/or repairing a tendon tear not at a point of boneattachment, in some embodiments, comprise biocompatible matricesincluding, but not limited to, fibrous collagen matrices, crosslinkedhyaluron, allograft tissue, other synthetic matrices or combinationsthereof.

In another aspect, the present invention provides a kit comprising asolution comprising PDGF in a first container and a second containercomprising a biocompatible matrix. In some embodiments, the solutioncomprises a predetermined concentration of PDGF. The concentration ofPDGF, in some embodiments, can be predetermined according to the natureof the tendon being treated. The kit may further comprise a scaffoldingmaterial and the scaffolding material may further comprise abiocompatible binder. Moreover, the amount of biocompatible matrixprovided by a kit can be dependent on the nature of the tendon beingtreated. Biocompatible matrix that may be included in the kit may be ascaffolding material, a scaffolding material and a biocompatible binder,and/or bone allograft such as DFDBA or particulate DBM. In oneembodiment the bone scaffolding material comprises a calcium phosphate,such as β-TCP. In another embodiment, a scaffolding material comprises atype I collagen patch as described herein. A syringe, in someembodiments, can facilitate disposition of the PDGF solution in thebiocompatible matrix for application at a surgical site, such as a siteof tendon attachment to bone. The kit may also contain instructions foruse.

Accordingly it is an object of the present invention to provide acomposition comprising PDGF useful in the attachment of tendon to bone.

Accordingly, it is an object of the present invention to provide acomposition comprising PDGF useful for repair of tendons.

It is another object of the present invention to provide a method forattachment of tendon to bone using a composition comprising PDGF.

Another object of the present invention is to provide a compositioncomprising PDGF and method of using this composition to attach rotatorcuff tendons to the humerus.

Another object of the present invention is to provide a compositioncomprising PDGF disposed in a matrix and a method of using thiscomposition to attach rotator cuff tendons to the humerus.

Another object of the present invention is to provide a compositioncomprising PDGF disposed in a matrix and further comprising a binder,and a method of using this composition to attach rotator cuff tendons tothe humerus

These and other embodiments of the present invention are described ingreater detail in the detailed description which follows. These andother objects, features and advantages of the present invention willbecome apparent after a review of the following detailed description ofthe disclosed embodiments and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a bone anchor according to an embodiment of thepresent invention.

FIG. 2 illustrates two embodiments, 30 and 36, of an encapsulated PDGFcomposition, also showing a PDGF pouch 32 and a suturing border 34.

FIGS. 3A, 3B and 3C illustrate a prior art technique (3A), a PDGFcontaining pad incorporated into sutures (3B), and a PDGF pouch suturedover a repaired tear (3C).

FIG. 4 illustrates a surgical procedure according to one embodiment ofthe present invention.

DETAILED DESCRIPTION

The present invention provides compositions and methods for thetreatment and/or repair of damaged tendons. In some embodiments,compositions and methods of the present invention are useful in theattachment or reattachment of tendons to bone, and may be applied to anytendon reattachment. In some embodiments, compositions and methods ofthe present invention enhance tendon attachment to bone by strengtheningthe tendon and/or bone at the site of tendon attachment to the bone.Moreover, the treatment of tendons encompasses application ofcompositions of the present invention to tendons, including damaged orinjured tendons, such as tendons exhibiting tearing, delamination,and/or any other strain or deformation. Tendons which may be reattachedto bone and/or treated by compositions and methods of the presentinvention include, but are not limited to, tendons of the subscapularis,supraspinatus, infraspinatus, teres minor, rectus femoris, tibialisposterior, and quadraceps femoris, as well as the Achilles Tendon,patellar tendon, abductor and adductor tendons, or other tendons of thehip.

The present invention, in one embodiment, for example, providescompositions and methods for the treatment of rotator cuff tears. Asused herein, rotator cuff tears include complete tendon detachment aswell as incomplete or partial tendon detachment. The presentcompositions and methods facilitate the healing response to rotator cuffrepairs and tendon reattachment to the humeral head.

In one embodiment, a composition for promoting tendon reattachment tobone, such as the humeral head, comprises a solution comprising PDGF anda biocompatible matrix, wherein the solution is disposed in thebiocompatible matrix. In another embodiment, a composition comprises aPDGF solution disposed in a biocompatible matrix, wherein thebiocompatible matrix comprises a bone scaffolding material and abiocompatible binder. In one aspect, biocompatible matrices may includecalcium phosphate particles with or without biocompatible binders orbone allograft such as DFDBA or particulate DBM. In another aspect,biocompatible matrices may include DFDBA or DBM.

Turning now to components that can be included in various embodiments ofthe present invention, compositions of the present invention comprise asolution comprising PDGF.

PDGF Solutions

In one aspect, a composition provided by the present invention comprisesa solution comprising platelet derived growth factor (PDGF) and abiocompatible matrix, wherein the solution is disposed in thebiocompatible matrix. In some embodiments, PDGF is present in thesolution in a concentration ranging from about 0.01 mg/ml to about 10mg/ml, from about 0.05 mg/ml to about 5 mg/ml, from about 0.1 mg/ml toabout 1.0 mg/ml. PDGF may be present in the solution at anyconcentration within these stated ranges. In other embodiments, PDGF ispresent in the solution at any one of the following concentrations:about 0.05 mg/ml; about 0.1 mg/ml; about 0.15 mg/ml; about 0.2 mg/ml;about 0.25 mg/ml; about 0.3 mg/ml; about 0.35 mg/ml; about 0.4 mg/ml;about 0.45 mg/ml; about 0.5 mg/ml, about 0.55 mg/ml, about 0.6 mg/ml,about 0.65 mg/ml, about 0.7 mg/ml; about 0.75 mg/ml; about 0.8 mg/ml;about 0.85 mg/ml; about 0.9 mg/ml; about 0.95 mg/ml; or about 1.0 mg/ml.It is to be understood that these concentrations are simply examples ofparticular embodiments, and that the concentration of PDGF may be withinany of the concentration ranges stated above.

Various amounts of PDGF may be used in the compositions of the presentinvention. Amounts of PDGF that could be used include amounts in thefollowing ranges: about 1 μg to about 50 mg, about 10 μg to about 25 mg,about 100 μg to about 10 mg, and about 250 μg to about 5 mg.

The concentration of PDGF or other growth factors in embodiments of thepresent invention can be determined by using an enzyme-linkedimmunoassay as described in U.S. Pat. Nos. 6,221,625, 5,747,273, and5,290,708, or any other assay known in the art for determining PDGFconcentration. When provided herein, the molar concentration of PDGF isdetermined based on the molecular weight of PDGF dimer (e.g., PDGF-BB;MW about 25 kDa).

In embodiments of the present invention, PDGF comprises PDGF homodimersand heterodimers, including PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD,and mixtures and derivatives thereof. In one embodiment, PDGF comprisesPDGF-BB. In another embodiment PDGF comprises a recombinant human PDGF,such as rhPDGF-BB.

PDGF, in some embodiments, can be obtained from natural sources. Inother embodiments, PDGF can be produced by recombinant DNA techniques.In other embodiments, PDGF or fragments thereof may be produced usingpeptide synthesis techniques known to one of skill in the art, such assolid phase peptide synthesis. When obtained from natural sources, PDGFcan be derived from biological fluids. Biological fluids, according tosome embodiments, can comprise any treated or untreated fluid associatedwith living organisms including blood

Biological fluids, in another embodiment, can also comprise bloodcomponents including platelet concentrate (PC), apheresed platelets,platelet-rich plasma (PRP), plasma, serum, fresh frozen plasma (FFP),and buffy coat (BC). Biological fluids, in a further embodiment, cancomprise platelets separated from plasma and resuspended in aphysiological fluid.

When produced by recombinant DNA techniques, a DNA sequence encoding asingle monomer (e.g., PDGF B-chain or A-chain), in some embodiments, canbe inserted into cultured prokaryotic or eukaryotic cells for expressionto subsequently produce the homodimer (e.g. PDGF-BB or PDGF-AA). Inother embodiments, a PDGF heterodimer can be generated by inserting DNAsequences encoding for both monomeric units of the heterodimer intocultured prokaryotic or eukaryotic cells and allowing the translatedmonomeric units to be processed by the cells to produce the heterodimer(e.g. PDGF-AB). Commercially available cGMP recombinant PDGF-BB can beobtained commercially from Chiron Corporation (Emeryville, Calif.).Research grade rhPDGF-BB can be obtained from multiple sources includingR&D Systems, Inc. (Minneapolis, Minn.), BD Biosciences (San Jose,Calif.), and Chemicon, International (Temecula, Calif.).

In embodiments of the present invention, PDGF comprises PDGF fragments.In one embodiment rhPDGF-B comprises the following fragments: amino acidsequences 1-31, 1-32, 33-108, 33-109, and/or 1-108 of the entire Bchain. The complete amino acid sequence (1-109) of the B chain of PDGFis provided in FIG. 15 of U.S. Pat. No. 5,516,896. It is to beunderstood that the rhPDGF compositions of the present invention maycomprise a combination of intact rhPDGF-B (1-109) and fragments thereof.Other fragments of PDGF may be employed such as those disclosed in U.S.Pat. No. 5,516,896. In accordance with one embodiment, the rhPDGF-BBcomprises at least 65% of intact rhPDGF-B (1-109). In accordance withother embodiments, the rhPDGF-BB comprises at least 75%, 80%, 85%, 90%,95%, or 99% of intact rhPDGF-B (1-109).

In some embodiments of the present invention, PDGF can be purified.Purified PDGF, as used herein, comprises compositions having greaterthan about 95% by weight PDGF prior to incorporation in solutions of thepresent invention. The solution may be any pharmaceutically acceptablesolution. In other embodiments, the PDGF can be substantially purified.Substantially purified PDGF, as used herein, comprises compositionshaving about 5% to about 95% by weight PDGF prior to incorporation intosolutions of the present invention. In one embodiment, substantiallypurified PDGF comprises compositions having about 65% to about 95% byweight PDGF prior to incorporation into solutions of the presentinvention. In other embodiments, substantially purified PDGF comprisescompositions having about 70% to about 95%, about 75% to about 95%,about 80% to about 95%, about 85% to about 95%, or about 90% to about95%, by weight PDGF, prior to incorporation into solutions of thepresent invention. Purified PDGF and substantially purified PDGF may beincorporated into scaffolds and binders.

In a further embodiment, PDGF can be partially purified. Partiallypurified PDGF, as used herein, comprises compositions having PDGF in thecontext of PRP, FFP, or any other blood product that requires collectionand separation to produce PDGF. Embodiments of the present inventioncontemplate that any of the PDGF isoforms provided herein, includinghomodimers and heterodimers, can be purified or partially purified.Compositions of the present invention containing PDGF mixtures maycontain PDGF isoforms or PDGF fragments in partially purifiedproportions. Partially purified and purified PDGF, in some embodiments,can be prepared as described in U.S. patent application Ser. No.11/159,533 (Publication No: 20060084602).

In some embodiments, solutions comprising PDGF are formed bysolubilizing PDGF in one or more buffers. Buffers suitable for use inPDGF solutions of the present invention can comprise, but are notlimited to, carbonates, phosphates (e.g. phosphate buffered saline),histidine, acetates (e.g. sodium acetate), acidic buffers such as aceticacid and HCl, and organic buffers such as lysine, Tris buffers (e.g.tris(hydroxymethyl)aminoethane),N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and3-(N-morpholino) propanesulfonic acid (MOPS). Buffers can be selectedbased on biocompatibility with PDGF and the buffer's ability to impedeundesirable protein modification. Buffers can additionally be selectedbased on compatibility with host tissues. In one embodiment, sodiumacetate buffer is used. The buffers may be employed at differentmolarities, for example about 0.1 mM to about 100 mM, about 1 mM toabout 50 mM, about 5 mM to about 40 mM, about 10 mM to about 30 mM, orabout 15 mM to about 25 mM, or any molarity within these ranges. In someembodiments, an acetate buffer is employed at a molarity of about 20 mM.

In another embodiment, solutions comprising PDGF are formed bysolubilizing lyophilized PDGF in water, wherein prior to solubilizationthe PDGF is lyophilized from an appropriate buffer.

Solutions comprising PDGF, according to embodiments of the presentinvention, can have a pH ranging from about 3.0 to about 8.0. In oneembodiment, a solution comprising PDGF has a pH ranging from about 5.0to about 8.0, more preferably about 5.5 to about 7.0, most preferablyabout 5.5 to about 6.5, or any value within these ranges.

The pH of solutions comprising PDGF, in some embodiments, can becompatible with the prolonged stability and efficacy of PDGF or anyother desired biologically active agent. PDGF is generally more stablein an acidic environment. Therefore, in accordance with one embodimentthe present invention comprises an acidic storage formulation of a PDGFsolution. In accordance with this embodiment, the PDGF solutionpreferably has a pH from about 3.0 to about 7.0, and more preferablyfrom about 4.0 to about 6.5. The biological activity of PDGF, however,can be optimized in a solution having a neutral pH range. Therefore, ina further embodiment, the present invention comprises a neutral pHformulation of a PDGF solution. In accordance with this embodiment, thePDGF solution preferably has a pH from about 5.0 to about 8.0, morepreferably about 5.5 to about 7.0, most preferably about 5.5 to about6.5. In accordance with a method of the present invention, an acidicPDGF solution is reformulated to a neutral pH composition, wherein suchcomposition is then used to treat bone, ligaments, tendons or cartilagein order to promote their growth and/or healing. In accordance with apreferred embodiment of the present invention, the PDGF utilized in thesolutions is rhPDGF-BB.

In some embodiments, the pH of the PDGF containing solution may bealtered to optimize the binding kinetics of PDGF to a matrix substrateor linker. If desired, as the pH of the material equilibrates toadjacent material, the bound PDGF may become labile.

The pH of solutions comprising PDGF, in some embodiments, can becontrolled by the buffers recited herein. Various proteins demonstratedifferent pH ranges in which they are stable. Protein stabilities areprimarily reflected by isoelectric points and charges on the proteins.The pH range can affect the conformational structure of a protein andthe susceptibility of a protein to proteolytic degradation, hydrolysis,oxidation, and other processes that can result in modification to thestructure and/or biological activity of the protein.

In some embodiments, solutions comprising PDGF can further compriseadditional components, such as other biologically active agents. Inother embodiments, solutions comprising PDGF can further comprise cellculture media, other stabilizing proteins such as albumin, antibacterialagents, protease inhibitors [e.g., ethylenediaminetetraacetic acid(EDTA), ethylene glycol-bis(beta-aminoethylether)-N, N,N′,N′-tetraaceticacid (EGTA), aprotinin, ε-aminocaproic acid (EACA), etc.] and/or othergrowth factors such as fibroblast growth factors (FGFs), epidermalgrowth factors (EGFs), transforming growth factors (TGFs), keratinocytegrowth factors (KGFs), insulin-like growth factors (IGFs), bonemorphogenetic proteins (BMPs), or other PDGFs including compositions ofPDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and/or PDGF-DD.

In addition to solutions comprising PDGF, compositions of the presentinvention also comprise a biocompatible matrix in which to dispose thePDGF solutions and may also comprise a biocompatible binder either withor without a biocompatible matrix.

Biocompatible Matrix

Scaffolding Material

A biocompatible matrix, according to embodiments of the presentinvention, comprises a scaffolding material. The scaffolding material,according to embodiments of the present invention, provides a frameworkor scaffold for new tissue growth to occur, including tendon and/or bonetissue. A scaffolding material, in some embodiments, comprises at leastone calcium phosphate. In other embodiments, a scaffolding material cancomprise a plurality of calcium phosphates. Calcium phosphates suitablefor use as a scaffolding material, in embodiments of the presentinvention, have a calcium to phosphorus atomic ratio ranging from 0.5 to2.0. In some embodiments the biocompatible matrix comprises an allograftsuch as DFDBA or particulate DBM.

Non-limiting examples of calcium phosphates suitable for use as bonescaffolding materials comprise amorphous calcium phosphate, monocalciumphosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA),dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous(DCPA), octacalcium phosphate (OCP), a-tricalcium phosphate, β-TCP,hydroxyapatite (OHAp), poorly crystalline hydroxyapatite, tetracalciumphosphate (TTCP), heptacalcium decaphosphate, calcium metaphosphate,calcium pyrophosphate dihydrate, carbonated calcium phosphate, andcalcium pyrophosphate.

Moreover, in some embodiments, a scaffolding material comprises acollagen patch or pad. A collagen patch or pad, in one embodiment of thepresent invention, comprises a fibrous collagen such as soluble type Ibovine collagen. Fibrous collagen suitable for use in collagen patchesor pads demonstrate sufficient mechanical properties, including wettensile strength, to withstand suturing and hold a suture withouttearing. In one embodiment, a collagen patch or pad has a densityranging from about 0.75 g/cm³ to about 1.5 g/cm³. Additionally, acollagen patch or pad for use in some embodiments of the presentinvention is porous and operable to absorb water in an amount rangingfrom about 1× to about 15× the mass of the collagen patch.

In some embodiments, a scaffolding material comprises porous structure.Porous bone scaffolding materials, according to some embodiments, cancomprise pores having diameters ranging from about 1 μm to about 1 mm.In one embodiment, a scaffolding material comprises macropores havingdiameters ranging from about 100 μm to about 1 mm. In anotherembodiment, a scaffolding material comprises mesopores having diametersranging from about 10 μm to about 100 μm. In a further embodiment, ascaffolding material comprises micropores having diameters less thanabout 10 μm. Embodiments of the present invention contemplatescaffolding materials comprising macropores, mesopores, and microporesor any combination thereof.

A porous scaffolding material, in one embodiment, has a porosity greaterthan about 25%. In another embodiment, a porous scaffolding material hasa porosity greater than about 50%. In a further embodiment, a porousscaffolding material has a porosity greater than about 90%.

In some embodiments, a scaffolding material comprises a plurality ofparticles. A scaffolding material, for example, can comprise a pluralityof calcium phosphate particles. Scaffolding particles, in oneembodiment, have an average diameter ranging from about 1 μm to about 5mm. In other embodiments, particles have an average diameter rangingfrom about 250 μm to about 750 μm. Scaffolding particles, in anotherembodiment, can have average diameter ranging from about 100 μm to about400 μm. In a further embodiment, the particles have an average diameterranging from about 75 μm to about 300 μm. In additional embodiments,scaffolding particles have an average diameter less than about 1 μm and,in some cases, less than about 1 mm.

Scaffolding materials, according to some embodiments, can be provided ina shape suitable for implantation (e.g., a sphere, a cylinder, or ablock). In other embodiments, bone scaffolding materials are moldable,extrudable, and/or injectable. Moldable scaffolding materials canfacilitate efficient placement of compositions of the present inventionin and around tendons and/or bone, including sites of tendon attachmentto bone. In some embodiments, moldable scaffolding materials are appliedto bone and/or tendons with a spatula or equivalent device. In someembodiments, scaffolding materials are flowable. Flowable scaffoldingmaterials, in some embodiments, can be applied to tendon reattachmentsites through a syringe and needle or cannula. In some embodiments, theflowable scaffolding materials can be applied to sites of tendonreattachment percutaneously. In other embodiments, flowable scaffoldingmaterials can be applied to a surgically exposed site of tendonreattachment. In a further embodiment, moldable and/or flowablescaffolding materials can be applied to bone anchors used in thereattachment of a tendon to a bone.

In some embodiments, scaffolding materials are bioresorbable. Ascaffolding material, in one embodiment, can be resorbed within one yearof in vivo implantation. In another embodiment, a scaffolding materialcan be resorbed within 1, 3, 6, or 9 months of in vivo implantation.Bioresorbability is dependent on: (1) the nature of the matrix material(i.e., its chemical make up, physical structure and size); (2) thelocation within the body in which the matrix is placed; (3) the amountof matrix material that is used; (4) the metabolic state of the patient(diabetic/non-diabetic, osteoporotic, smoker, old age, steroid use,etc.); (5) the extent and/or type of injury treated; and (6) the use ofother materials in addition to the matrix such as other bone anabolic,catabolic and anti-catabolic factors.

Scaffolding Comprising β-Tricalcium Phosphate (β-TCP) A scaffoldingmaterial for use as a biocompatible matrix, in some embodiments,comprises β-TCP. β-TCP, according to some embodiments, can comprise aporous structure having multidirectional and interconnected pores ofvarying diameters. In some embodiments, β-TCP comprises a plurality ofpockets and non-interconnected pores of various diameters in addition tothe interconnected pores. The porous structure of β-TCP, in oneembodiment, comprises macropores having diameters ranging from about 100μm to about 1 mm, mesopores having diameters ranging from about 10 μm toabout 100 μm, and micropores having diameters less than about 10 μm.Macropores and micropores of the β-TCP can facilitate tissue in-growthincluding osteoinduction and osteoconduction while macropores, mesoporesand micropores can permit fluid communication and nutrient transport tosupport tissue and bone regrowth throughout the β-TCP biocompatiblematrix.

In comprising a porous structure, β-TCP, in some embodiments, can have aporosity greater than 25%. In other embodiments, β-TCP can have aporosity greater than 50%. In a further embodiment, β-TCP can have aporosity greater than 90%.

In some embodiments, a bone scaffolding material comprises β-TCPparticles. β-TCP particles, in one embodiment, have an average diameterranging from about 1 μm to about 5 mm. In other embodiments, β-TCPparticles have an average diameter ranging from about 250 μm to about750 μm. In another embodiment, β-TCP particles have an average diameterranging from about 100 μm to about 400 μm. In a further embodiment,β-TCP particles have an average diameter ranging from about 75 μm toabout 300 μm. In additional embodiments, β-TCP particles have an averagediameter less than 25 μm and, in some cases, sizes less than 1 mm.

A biocompatible matrix comprising a β-TCP scaffolding material, in someembodiments, is provided in a shape suitable for implantation (e.g., asphere, a cylinder, or a block). In other embodiments, a β-TCPscaffolding material is moldable, extrudable, and/or flowable therebyfacilitating application of the matrix in areas of tendon reattachment,such as channels in the humeral head. Flowable matrices may be appliedthrough syringes, tubes, or spatulas. In some embodiments, moldable,extrudable, and/or flowable β-TCP scaffolding materials are applied tobone anchors used in the reattachment of tendons to bone.

A β-TCP scaffolding material, according to some embodiments, isbioresorbable. In one embodiment, a β-TCP scaffolding material can be atleast 75% resorbed one year subsequent to in vivo implantation. Inanother embodiment, a β-TCP bone scaffolding material can be greaterthan 90% resorbed one year subsequent to in vivo implantation.

Scaffolding Material Comprising a Collagen Patch

In some embodiments, a scaffolding material comprises a collagen patchor pad. A collagen patch or pad, in one embodiment of the presentinvention, comprises a fibrous collagen such as soluble type I bovinecollagen. In another embodiment, a fibrous collagen comprises type II ortype III collagen. Fibrous collagen suitable for use in collagen patchesor pads demonstrate sufficient mechanical properties, including wettensile strength, to withstand suturing and hold a suture withouttearing. A fibrous collagen patch, for example, can have a wet tearstrength ranging from about 0.75 pounds to about 5 pounds. In oneembodiment, a collagen patch or pad has a density ranging from about0.75 g/cm³ to about 1.5 g/cm³. Additionally, a collagen patch or pad foruse in some embodiments of the present invention is porous and operableto absorb water in an amount ranging from about 1× to about 15× the massof the collagen patch.

Scaffolding Material and Biocompatible Binder

In another embodiment, a biocompatible matrix comprises a scaffoldingmaterial and a biocompatible binder. Biocompatible matrices comprising ascaffolding material and a biocompatible binder, according toembodiments of the present invention, are useful for the repair,strengthening, and/or reattachment of tendons to bone by providing astructure for new tendon and/or bone tissue growth.

Biocompatible binders, according to some embodiments, can comprisematerials operable to promote cohesion between combined substances. Abiocompatible binder, for example, can promote adhesion betweenparticles of a bone scaffolding material in the formation of abiocompatible matrix. In certain embodiments, the same material mayserve as both a scaffolding material and a binder if such material actsto promote cohesion between the combined substances and provides aframework for new tissue growth to occur, including tendon and bonegrowth.

Biocompatible binders, in some embodiments, can comprise collagen,elastin, polysaccharides, nucleic acids, carbohydrates, proteins,polypeptides, poly(α-hydroxy acids), poly(lactones), poly(amino acids),poly(anhydrides), polyurethanes, poly(orthoesters),poly(anhydride-co-imides), poly(orthocarbonates), poly(α-hydroxyalkanoates), poly(dioxanones), poly(phosphoesters), polylactic acid,poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(L-lactide-co-D, L-lactide),poly(D,L-lactide-co-trimethylene carbonate), polyglycolic acid,polyhydroxybutyrate (PHB), poly(ε-caprolactone), poly(δ-valerolactone),poly(γ-butyrolactone), poly(caprolactone), polyacrylic acid,polycarboxylic acid, poly(allylamine hydrochloride),poly(diallyldimethylammonium chloride), poly(ethyleneimine),polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone,polyethylene, polymethylmethacrylate, carbon fibers, poly(ethyleneglycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-polypropylene oxide) block copolymers, poly(ethyleneterephthalate)polyamide, and copolymers and mixtures thereof.

Biocompatible binders, in other embodiments, can comprise alginic acid,arabic gum, guar gum, xantham gum, gelatin, chitin, chitosan, chitosanacetate, chitosan lactate, chondroitin sulfate, N,O-carboxymethylchitosan, a dextran (e.g., α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, or sodium dextran sulfate), fibrin glue, lecithin,phosphatidylcholine derivatives, glycerol, hyaluronic acid, sodiumhyaluronate, a cellulose (e.g., methylcellulose, carboxymethylcellulose,hydroxypropyl methylcellulose, or hydroxyethyl cellulose), aglucosamine, a proteoglycan, a starch (e.g., hydroxyethyl starch orstarch soluble), lactic acid, a pluronic acids, sodium glycerophosphate,glycogen, a keratin, silk, and derivatives and mixtures thereof.

In some embodiments, a biocompatible binder is water-soluble. Awater-soluble binder can dissolve from the biocompatible matrix shortlyafter its implantation, thereby introducing macroporosity into thebiocompatible matrix. Macroporosity, as discussed herein, can increasethe osteoconductivity of the implant material by enhancing the accessand, consequently, the remodeling activity of the osteoclasts andosteoblasts at the implant site.

In some embodiments, a biocompatible binder can be present in abiocompatible matrix in an amount ranging from about 5 weight percent toabout 50 weight percent of the matrix. In other embodiments, abiocompatible binder can be present in an amount ranging from about 10weight percent to about 40 weight percent of the biocompatible matrix.In another embodiment, a biocompatible binder can be present in anamount ranging from about 15 weight percent to about 35 weight percentof the biocompatible matrix. In a further embodiment, a biocompatiblebinder can be present in an amount of about 20 weight percent of thebiocompatible matrix.

A biocompatible matrix comprising a scaffolding material and abiocompatible binder, according to some embodiments, can be flowable,moldable, and/or extrudable. In such embodiments, a biocompatible matrixcan be in the form of a paste or putty. A biocompatible matrix in theform of a paste or putty, in one embodiment, can comprise particles of ascaffolding material adhered to one another by a biocompatible binder.

A biocompatible matrix in paste or putty form can be molded into thedesired implant shape or can be molded to the contours of theimplantation site. In one embodiment, a biocompatible matrix in paste orputty form can be injected into an implantation site with a syringe orcannula. In a further embodiment, moldable and/or flowable scaffoldingmaterials can be applied to bone anchors used in the reattachment of atendon to a bone.

In some embodiments, a biocompatible matrix in paste or putty form doesnot harden and retains a flowable and moldable form subsequent toimplantation. In other embodiments, a paste or putty can hardensubsequent to implantation, thereby reducing matrix flowability andmoldability.

A biocompatible matrix comprising a scaffolding material and abiocompatible binder, in some embodiments, can also be provided in apredetermined shape including a block, sphere, or cylinder or anydesired shape, for example a shape defined by a mold or a site ofapplication.

A biocompatible matrix comprising a scaffolding material and abiocompatible binder, in some embodiments, is bioresorbable. Abiocompatible matrix, in such embodiments, can be resorbed within oneyear of in vivo implantation. In another embodiment, a biocompatiblematrix comprising a bone scaffolding material and a biocompatible bindercan be resorbed within 1, 3, 6, or 9 months of in vivo implantation.Bioresorbablity, in some embodiments, is dependent on: (1) the nature ofthe matrix material (i.e., its chemical make up, physical structure andsize); (2) the location within the body in which the matrix is placed;(3) the amount of matrix material that is used; (4) the metabolic stateof the patient (diabetic/non-diabetic, osteoporotic, smoker, old age,steroid use, etc.); (5) the extent and/or type of injury treated; and(6) the use of other materials in addition to the matrix such as otherbone anabolic, catabolic and anti-catabolic factors.

Biocompatible Matrix Comprising β-TCP and Collagen

In some embodiments, a biocompatible matrix can comprise a β-TCPscaffolding material and a biocompatible collagen binder. β-TCPscaffolding materials suitable for combination with a collagen binderare consistent with those provided hereinabove.

A collagen binder, in some embodiments, comprises any type of collagen,including Type I, Type II, and Type III collagens. In one embodiment, acollagen binder comprises a mixture of collagens, such as a mixture ofType I and Type II collagen. In other embodiments, a collagen binder issoluble under physiological conditions. Other types of collagen presentin bone or musculoskeletal tissues may be employed. Recombinant,synthetic and naturally occurring forms of collagen may be used in thepresent invention.

A biocompatible matrix, according to some embodiments, comprises aplurality of β-TCP particles adhered to one another with a collagenbinder. In one embodiment, β-TCP particles suitable for combination witha collagen binder have an average diameter ranging from about 1 μm toabout 5 mm. In another embodiment, β-TCP particles suitable forcombination with a collagen binder have an average diameter ranging fromabout 1 μm to about 1 mm. In other embodiments, β-TCP particles have anaverage diameter ranging from about 200 μm to about 3 mm or about 200 μmto about 1 mm, or about 1 mm to about 2 mm. In some embodiments, β-TCPparticles have an average diameter ranging from about 250 μm to about750 μm. β-TCP particles, in other embodiments, have an average diameterranging from about 100 μm to about 400 μm. In a further embodiment,β-TCP particles have an average diameter ranging from about 75 μm toabout 300 μm. In additional embodiments, β-TCP particles have an averagediameter less than about 25 μm and, in some cases, less than about 1 mm.

β-TCP particles, in some embodiments, can be adhered to one another bythe collagen binder so as to produce a biocompatible matrix having aporous structure. In some embodiments, a biocompatible matrix comprisingβ-TCP particles and a collagen binder can comprise pores havingdiameters ranging from about 1 μm to about 1 mm. A biocompatible matrixcomprising β-TCP particles and a collagen binder can comprise macroporeshaving diameters ranging from about 100 μm to about 1 mm, mesoporeshaving diameters ranging from about 10 μm to 100 μm, and microporeshaving diameters less than about 10 μm.

A biocompatible matrix comprising β-TCP particles and a collagen bindercan have a porosity greater than about 25%. In another embodiment, thebiocompatible matrix can have a porosity greater than about 50%. In afurther embodiment, the biocompatible matrix can have a porosity greaterthan about 90%.

A biocompatible matrix comprising β-TCP particles, in some embodiments,can comprise a collagen binder in an amount ranging from about 5 weightpercent to about 50 weight percent of the matrix. In other embodiments,a collagen binder can be present in an amount ranging from about 10weight percent to about 40 weight percent of the biocompatible matrix.In another embodiment, a collagen binder can be present in an amountranging from about 15 weight percent to about 35 weight percent of thebiocompatible matrix. In a further embodiment, a collagen binder can bepresent in an amount of about 20 weight percent of the biocompatiblematrix.

A biocompatible matrix comprising β-TCP particles and a collagen binder,according to some embodiments, can be flowable, moldable, and/orextrudable. In such embodiments, the biocompatible matrix can be in theform of a paste or putty. A paste or putty can be molded into thedesired implant shape or can be molded to the contours of theimplantation site. In one embodiment, a biocompatible matrix in paste orputty form comprising β-TCP particles and a collagen binder can beinjected into an implantation site with a syringe or cannula. In afurther embodiment, moldable, extrudable, and/or flowable matrixcomprising β-TCP particles and a collagen binder can be applied to boneanchors and/or sutures used in the reattachment of a tendon to a bone.

In some embodiments, a biocompatible matrix in paste or putty formcomprising β-TCP particles and a collagen binder can retain a flowableand moldable form when implanted. In other embodiments, the paste orputty can harden subsequent to implantation, thereby reducing matrixflowability and moldability.

A biocompatible matrix comprising β-TCP particles and a collagen binder,in some embodiments, can be provided in a predetermined shape such as ablock, sphere, or cylinder.

A biocompatible matrix comprising β-TCP particles and a collagen bindercan be resorbable. In one embodiment, a biocompatible matrix comprisingβ-TCP particles and a collagen binder can be at least 75% resorbed oneyear subsequent to in vivo implantation. In another embodiment, abiocompatible matrix comprising β-TCP particles and a collagen bindercan be greater than 90% resorbed one year subsequent to in vivoimplantation.

In some embodiments, a solution comprising PDGF can be disposed in abiocompatible matrix to produce a composition for the treatment ofrotator cuff tears.

Disposing PDGF Solution in a Biocompatible Matrix

In another aspect, the present invention provides methods for producingcompositions for use in the treatment of damaged or injured tendons,including those associated with torn rotator cuffs. In one embodiment, amethod for producing such compositions for the treatment of tendonsand/or bone comprises providing a solution comprising PDGF, providing abiocompatible matrix, and disposing the solution in the biocompatiblematrix. PDGF solutions and biocompatible matrices suitable forcombination are consistent with those described hereinabove.

In some embodiments, a PDGF solution can be disposed in a biocompatiblematrix by soaking the biocompatible matrix in the PDGF solution. A PDGFsolution, in another embodiment, can be disposed in a biocompatiblematrix by injecting the biocompatible matrix with the PDGF solution. Insome embodiments, injecting a PDGF solution can comprise disposing thePDGF solution in a syringe and expelling the PDGF solution into thebiocompatible matrix to saturate the biocompatible matrix.

The biocompatible matrix, according to some embodiments, can be in apredetermined shape, such as a brick or cylinder, prior to receiving aPDGF solution. Subsequent to receiving a PDGF solution, thebiocompatible matrix can have a paste or putty form that is flowable,extrudable, and/or injectable. In other embodiments, the biocompatiblematrix can already demonstrate a flowable paste or putty form prior toreceiving a solution comprising PDGF.

Compositions Further Comprising Biologically Active Agents

Compositions of the present invention, according to some embodiments,further comprise one or more biologically active agents in addition toPDGF. Biologically active agents that can be incorporated intocompositions of the present invention, in addition to PDGF, can compriseorganic molecules, inorganic materials, proteins, peptides, nucleicacids (e.g., genes, gene fragments, small-insert ribonucleic acids[si-RNAs], gene regulatory sequences, nuclear transcriptional factorsand antisense molecules), nucleoproteins, polysaccharides (e.g.,heparin), glycoproteins, and lipoproteins. Non-limiting examples ofbiologically active compounds that can be incorporated into compositionsof the present invention, including, e.g., anti-cancer agents,antibiotics, analgesics, anti-inflammatory agents, immunosuppressants,enzyme inhibitors, antihistamines, hormones, muscle relaxants,prostaglandins, trophic factors, osteoinductive proteins, growthfactors, and vaccines, are disclosed in U.S. patent application Ser. No.11/159,533 (Publication No: 20060084602). Biologically active compoundsthat can be incorporated into compositions of the present invention, insome embodiments, include osteoinductive factors such as insulin-likegrowth factors, fibroblast growth factors, or other PDGFs. In accordancewith other embodiments, biologically active compounds that can beincorporated into compositions of the present invention preferablyinclude osteoinductive and osteostimulatory factors such as bonemorphogenetic proteins (BMPs), BMP mimetics, calcitonin, calcitoninmimetics, statins, statin derivatives, fibroblast growth factors,insulin-like growth factors, growth differentiating factors, and/orparathyroid hormone. Additional factors for incorporation intocompositions of the present invention, in some embodiments, includeprotease inhibitors, as well as osteoporotic treatments that decreasebone resorption including bisphosphonates, and antibodies to the NF-kB(RANK) ligand.

Standard protocols and regimens for delivery of additional biologicallyactive agents are known in the art. Additional biologically activeagents can introduced into compositions of the present invention inamounts that allow delivery of an appropriate dosage of the agent to thedamaged tendon and/or the site of tendon reattachment. In most cases,dosages are determined using guidelines known to practitioners andapplicable to the particular agent in question. The amount of anadditional biologically active agent to be included in a composition ofthe present invention can depend on such variables as the type andextent of the condition, the overall health status of the particularpatient, the formulation of the biologically active agent, releasekinetics, and the bioresorbability of the biocompatible matrix. Standardclinical trials may be used to optimize the dose and dosing frequencyfor any particular additional biologically active agent.

A composition for the treatment of tendons and/or bone, according tosome embodiments, further comprises other bone grafting materials withPDGF including autologous bone marrow, autologous platelet extracts,allografts, synthetic bone matrix materials, xenografts, and derivativesthereof.

Methods of Treating and Reattaching Tendons

The present invention also provides methods for the attachment orreattachment of tendons to bone, the strengthening of tendon attachmentto bone as well as the treatment of tendons, such as tendons exhibitingtearing, delamination, or any other strain or deformation. In oneembodiment, a method for reattaching a tendon to bone comprisesproviding a composition comprising a PDGF solution disposed in abiocompatible matrix and applying the composition to at least one siteof tendon reattachment on the bone. In another embodiment, a method ofstrengthening the attachment of a tendon to a bone comprises providing acomposition comprising a PDGF solution disposed in a biocompatiblematrix and applying the composition to at least one site of tendonattachment to bone. Methods of strengthening tendon attachment to bone,in some embodiments, assist in preventing or inhibiting tendondetachment from bone, such as in rotator cuff injuries.

The present invention also provides methods of treating rotator cufftears. In one embodiment, a method for treating rotator cuff tearscomprises providing a composition comprising a PDGF solution disposed ina biocompatible matrix and applying the composition to at least one siteof tendon reattachment on the humeral head. In some embodiments,applying the composition to at least one site of tendon reattachment cancomprise molding the composition to the contours of the reattachmentsite on the humeral head. A composition, for example, can be molded intoa channel formed on a surface of the humeral head for receiving thedetached tendon. The composition may be applied to the vicinity of theinsertion site of the tendon into bone to further strengthen theattachment.

In some embodiments, a method for treating rotator cuff tears furthercomprises disposing at least one anchoring means, such as a bone anchorin the humeral head, wherein the bone anchor further comprises a PDGFcomposition, and coupling at least one detached tendon to the boneanchor. In embodiments of the present invention, tendons can be securedto bone anchors through sutures. Sutures may also be soaked in PDGFsolutions or coated in PDGF compositions before use. Examples 2-4describe three different methods for treating rotator cuff tears.

In another embodiment, a method of treating a tendon comprises providinga composition comprising a PDGF solution disposed in a biocompatiblematrix and applying the composition to a surface of at least one tendon.In some embodiments, the at least one tendon is an injured or damagedtendon, such as tendon exhibiting tearing, delamination, or any otherdeformation.

PDGF solutions and biocompatible matrices suitable for use incompositions, according to embodiments of methods of the presentinvention, are consistent with those provided hereinabove.

Kits

In another aspect, the present invention provides a kit comprising asolution comprising PDGF in a first container and a second containercomprising a biocompatible matrix. In some embodiments, the solutioncomprises a predetermined concentration of PDGF. The concentration ofPDGF, in some embodiments, can be predetermined according to the natureof the tendon being treated. The kit may further comprise a scaffoldingmaterial and the scaffolding material may further comprise abiocompatible binder. Moreover, the amount of biocompatible matrixprovided by a kit can be dependent on the nature of the tendon beingtreated. Biocompatible matrix that may be included in the kit may be ascaffolding material, a scaffolding material and a biocompatible binder,and/or bone allograft such as DFDBA or particulate DBM. In oneembodiment the bone scaffolding material comprises a calcium phosphate,such as β-TCP. In another embodiment, a scaffolding material comprises atype I collagen patch as described herein. A syringe, in someembodiments, can facilitate disposition of the PDGF solution in thebiocompatible matrix for application at a surgical site, such as a siteof tendon attachment to bone. The kit may also contain instructions foruse.

The following examples will serve to further illustrate the presentinvention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various embodiments, modifications and equivalentsthereof which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the invention.

Example 1 Preparation of a Composition Comprising a Solution of PDGF anda Biocompatible Matrix

A composition comprising a solution of PDGF and a biocompatible matrixwas prepared according to the following procedure.

A pre-weighed block of biocompatible matrix comprising β-TCP andcollagen was obtained. The β-TCP comprised pure β-TCP particles havingsizes ranging from about 75 μm to about 300 μm. The β-TCP particles wereformulated with approximately 20% weight percent soluble bovine collagenbinder. A β-TCP/collagen biocompatible matrix can be commerciallyobtained from Kensey Nash (Exton, Pa.).

A solution comprising rhPDGF-BB was obtained. rhPDGF-BB is commerciallyavailable from Chiron Corporation at a stock concentration of 10 mg/ml(i.e., Lot # QA2217) in a sodium acetate buffer. The rhPDGF-BB isproduced in a yeast expression system by Chiron Corporation and isderived from the same production facility as the rhPDGF-BB that isutilized in the products REGRANEX, (Johnson & Johnson) and GEM 21S(BioMimetic Therapeutics) which has been approved for human use by theUnited States Food and Drug Administration. This rhPDGF-BB is alsoapproved for human use in the European Union and Canada. The rhPDGF-BBsolution was diluted to 0.3 mg/ml in the acetate buffer. The rhPDGF-BBsolution can be diluted to any desired concentration according toembodiments of the present invention.

A ratio of about 91 μl of rhPDGF-BB solution to about 100 mg dry weightof the β-TCP/collagen biocompatible matrix was used to produce thecomposition. The rhPDGF-BB solution was expelled on the biocompatiblematrix with a syringe, and the resulting composition was blended andmolded into a thin strand for insertion into a 1 cc tuberculin syringefor placement at a site of tendon reattachment.

Example 2 Treating Rotator Cuff Tears with an Open Repair Method

Open repair is performed without arthroscopy, and is typically used forlarger rotator cuff injuries. In accordance with this method of thepresent invention, a surgeon makes a two- to three-inch incision overthe shoulder and separates the deltoid muscle from the anterior acromionto gain access to and improve visualization of the torn rotator cuff.The deltoid muscle should only be detached to the extent necessary togain sufficient access to the rotator cuff injury. Following the rotatorcuff repair procedure, the deltoid is repaired with sutures to close thelongitudinal divisional of the muscle.

The surgeon then identifies the detached end of the involved tendon(s)(infraspinatus, supraspinatus, teres minor, and/or subscapularis) andthe remaining tendon stump is cut away or removed from the humeral headpreferably with a rasp, rongeur, scalpel or high speed bur and/orshaver. The surgeon may perform an acromioplasty (removal of bone spursfrom the undersurface of the acromion) and remove any scar tissue thathas built up on the tendon. Following debridement, cortical bone of thehumeral head is abraded so as to produce bleeding bone and provideaccess for migrating mesenchymal stem cells of the bone marrow. Inaccordance with one embodiment of the method of the present invention,the cortical bone is removed so as to form a small channel in thehumeral head that corresponds in shape and size to the original tendonattachment footprint. Preferably the channel is formed adjacent to thearticular cartilage of the shoulder joint.

Prior to reattachment of the tendon, the surgeon may drill small holeswithin the channel through the bone. These holes may be used to affixbone anchors (preferably bone anchor screws). The bone anchors may beformed from any biocompatible material, and are preferably made ofeither a biocompatible metal or a resorbable composition. In accordancewith an embodiment of the invention, bone anchor screws are affixed in adouble row arrangement. The anchors thereby become an attachment pointused to affix sutures to the humeral head.

A pilot hole is first drilled prior to insertion of the anchors. A PDGFcomposition in accordance with the present invention is then put intothe pilot hole prior to insertion of the anchor. In one embodiment,injectable forms of the PDGF composition of the present invention areinjected into the pilot holes.

As shown in FIG. 1, in an alternative embodiment, a self-tappingself-drilling cannulated anchor 10 is used without the use of an initialpilot hole. Anchor 10 includes a needle access port 12 at or near itsproximal end 14, a central channel 16 extending along the axis of anchor10, and one or more exit ports, including radial exit ports 18 along theaxis of anchor 10, and/or a distal exit port 20 located near the distalend 22 of anchor 10. In one embodiment, anchor 10 is drilled into thechannel. Preferably a plurality of anchors 10 are used. Once insertedinto the humeral head, a needle is inserted into the needle access port12 of anchor 10, and a PDGF composition of the present invention isinjected into the central channel 16. A sufficient amount of PDGFcomposition is injected into the anchor such that the PDGF compositionfills the central channel 16 and flows out of the radial exit ports 18and/or distal exit port 20 and into the surrounding bone. Any effectiveamount or concentration of PDGF composition may be used. In oneembodiment, approximately 0.1 to 1.0 cc of a composition havingapproximately 0.3 to 1 mg/ml of PDGF is injected into each anchor orpilot hole.

In accordance with an embodiment of the present invention, the exitports included in anchor 10 may be of varying diameter in order toregulate the rate at which the PDGF migrates into the surrounding bone.In addition, the rate of PDGF release within the surrounding bone isregulated by utilizing different PDGF formulations in the variousanchors inserted into the channel. For example, the rate of PDGF releaseis prolonged by using more viscous compositions in certain anchors, orby using PDGF compositions comprising a matrix with extended PDGFrelease characteristics.

Alternatively, the drilled holes are used to affix sutures directly tothe humeral head without the use of bone anchors.

In accordance with the next step of the method of the present invention,a PDGF composition is applied to substantially cover the channel. ThePDGF composition used to cover the channel is in the form of either asolution, a putty, or gel, as described herein above. Alternatively, thePDGF composition is in the form of a pad. The pad may be composed of asubstrate that is hydrated with a PDGF solution. The substrate is madefrom fibrous type I collagen, collagen hydrogel, crosslinked hyaluronicacid, porcine small intestine submucosa (SIS), polylacticacid/polyglycolic acid, or cellulose.

After the channel is covered with the PDGF composition, the proximal endof the tendon is then placed over the PDGF composition and into thechannel. The tendon is secured in place by use of sutures that passthrough tendon, the PDGF composition and into bone or through theeyelets of the bone anchors. Any of the various standard suturingtechniques known to those skilled in the art (e.g., Mason Allen,mattress, simple suturing) may be used.

In accordance with an embodiment, the sutures are also impregnated witha PDGF solution prior to use. The sutures may be soaked in or saturatedwith a PDGF composition. Any effective amount or concentration of PDGFcomposition may be used. In one embodiment, PDGF at concentrations of0.1, 0.3, or 1.0 mg/mL may be used to wet the suture prior to use.Furthermore, the suture may be treated with glycerol, gelatin, orparaffin wax to slow the release of PDGF in a manner that is consistentwith the wound healing process.

The PDGF composition may be applied adjacent to and/or over the tendonto augment healing of the tendon/bone margins. This PDGF composition maybe in the form of a solution, putty, gel, or pad, and may be secured inposition with the same sutures used to secure the tendon.

Following the implantation of the PDGF composition and suturing of therotator cuff, all dissected muscles are sutured closed, the overlyingfascia is repaired, and lastly the patient's skin is closed with suturesor staples.

Example 3 Treating Rotator Cuff Tears with a Mini-Open Repair Method

A mini-open rotator cuff repair procedure involves using both anarthroscopic technique for part of the process in conjunction with alimited open technique typically done through a 3 cm to 5 cm incision.This technique also incorporates an arthroscopy to visualize the tear,assess and treat damage to other structures within the joint (i.e.,labrum and remove the spurs under the acromion). Arthroscopic removal ofspurs (acromioplasty) avoids the need to detach the deltoid muscle.Thereafter, an arthroscopic decompression may be performed. Thedecompression may be followed by a release and mobilization of thetendons and placement of tagging sutures. These steps may be donearthroscopically or open. The final steps are done in an open procedure,but via the smaller opening. In particular, a small lateral deltoidsplit is performed in order to place tendon-gripping sutures on thepreviously mobilized cuff and to fix the cuff to bone using eithersuture anchors or transosseous sutures.

In accordance with one embodiment, a mini-open repair method of thepresent invention comprises the following steps.

The patient is prepared in accordance with standard techniques forpatient positioning and marking. The arthroscope is placed in theglenohumeral joint through the posterior portal and a thoroughevaluation of the joint is performed. The rotator cuff tear isidentified and a lateral portal is created.

Rotator cuff mobilization starts with an intra-articular release. Ahooked electrocautery device is used to release the cuff from theglenoid labrum. This allows mobilization of the entire cuff if necessary(anterior to posterior). Once the intra-articular release has beenperformed, the arthroscope is directed to the subacromial space.

An arthroscopic subacromial bursectomy is performed. The tuberosity(area of rotator cuff insertion) is decorticated. In some applicationsthe tuberosity may be only slightly decorticate with no formal channelbeing created. A shaver is used to debride any of the torn cuff edgethat appears to be nonviable or attenuated. Stay sutures are placed inthe edge of the cuff tear approximately 1 cm apart. The stay sutures maybe pretreated with a PDGF composition in the manner described above inExample 2. Additional releases of the cuff from the glenoid areperformed as necessary.

The mini-open approach is then initiated with a horizontal lateralincision (3-4 cm long) being made over the lateral edge of the acromion.The deltoid muscle fibers are split to expose the rotator cuff tear.

If the tear is small and easily mobilized, sutures are placed throughthe edge of the cuff tear which is then repaired using suture anchorsplaced in the superolateral aspect of the greater tuberosity. For largetears under some tension, special intratendinous sutures are placedthrough the cuff and these are then repaired using the suture anchorsplaced in the superolateral greater tuberosity. Prior to securing thecuff, a PDGF composition is placed between the tendon and the humeralhead in the same manner as described above regarding open procedures. Inaddition, the sutures used to secure the tendon may be pretreated withPDGF in a manner similar to that described above. The suture anchors maybe of the type described above and illustrated in FIG. 1. A PDGFcomposition may be injected into the suture anchors or into the sutureanchor holes as described above. The surgery is completed in accordancewith known closure techniques.

This arthroscopically assisted open repair has limitations when dealingwith large or massive rotator cuff repairs. The necessary surgicalreleases can be difficult, if not impossible, to perform through a smalltrans-deltoid split. When compared to complete arthroscopic repair, themini-open repair provides more secure bone-to-tendon fixation sincetendon gripping sutures can be used.

Example 4 Treating Rotator Cuff Tears with an All-Arthroscopic RepairMethod

This technique uses multiple small incisions (portals) and arthroscopictechnology to visualize and repair the rotator cuff. In addition, inaccordance with the present invention this technique utilizes injectableor small encapsulated PDGF compositions that are capable of insertionthrough a keyhole incision or a cannula so that they are amenable to usewith arthroscopic techniques.

In accordance with one embodiment, an arthroscopic repair method of thepresent invention comprises the following steps. The patient is preparedin accordance with standard techniques for patient positioning,assessment and marking. One or two very small (1 cm) incisions, or“portals” are made, preferably one in the front and one behind theshoulder joint. Through these small portals, hollow instruments calledcannulae are placed that irrigate the inside of the shoulder joint withsterile saline and inflate the joint with clear fluid. The cannulaeallow the placement of an arthroscopic camera and specially designedinstruments within the shoulder joint. The surgeon inserts a camera intothe joint and maneuvers the camera around the joint in order to performdiagnostic arthroscopy.

In the most common cases the diagnostic arthroscopy reveals that thesupraspinatus tendon is torn and/or pulled back slightly from its normalattachment at the greater tuberosity of the humerus. These smaller tearswhich are non-retracted or minimally-retracted only need to be freshenedor debrided back to stable, healthy tendon tissue, then mobilized backto the tuberosity and fixed in place. The surgeon utilizes sutureanchors to hold the tear in position while it heals. As with anchorsused in the procedures described in Example 2, these anchors can be madeof metal or absorbable compounds. In addition, the anchors are bescrewed or pressed into the bone of the attachment site and the attachedsutures used to tie the edge of the rotator cuff in place.

Prior to securing the tendon, a protected PDGF composition is placedbetween the tendon and the humeral head. The material may be placed inthe bone anchor holes as well as across the decorticated surface of thehumeral head. In the event that the humeral head is not prepared bydecortication, the PDGF composition is placed against the cortical boneof the humeral head, and the tendon sutured into place in a standardmanner. The sutures used to secure the tendon may be impregnated withPDGF in a manner similar to that described above. The suture anchors maybe of the type described above and illustrated in FIG. 1. An injectablePDGF composition may be injected into the suture anchors by inserting aneedle through one of the cannula.

As tears become larger, they deform and the tendon tissue shrinks Thus,larger tears need to be refashioned, repaired side-to-side, or zippedclosed using a technique called margin convergence. This technique isanalogous to zippering shut an open tent flap. The rotator cuff tissueis freed from a scarred, retracted position. A protected PDGFcomposition is then inserted through one of the cannula or directlythrough an incision. The protected PDGF composition comprises a PDGFcomposition as described herein above encapsulated in or otherwiseassociated with a membrane designed to protect the PDGF composition fromthe arthroscopic fluid environment of the surgical site (FIG. 3). ThePDGF can be released at the treatment site using a variety of techniquesto protect the protein during the initial placement to avoid rapid lossfrom the site due to the use of high volumes of fluids associated withthe surgical procedure. In one embodiment, a membrane displays anintrinsic charge that promotes ionic interactions operable to releasethe PDGF in response to changing ionic conditions at the treatment orreattachment site. In another embodiment, a membrane forms a covalentinteraction with the PDGF that is reversible via hydrolysis or enzymaticdigestion to release the PDGF at the reattachment or treatment site.Membranes for protecting PDGF, in some embodiments, comprise collagen,polysaccharides, nucleic acids, carbohydrates, proteins, polypeptides,poly(α-hydroxy acids), poly(lactones), poly(amino acids),poly(anhydrides), poly(orthoesters), poly(anhydride-co-imides),poly(orthocarbonates), poly(α-hydroxy alkanoates), poly(dioxanones),poly(phosphoesters), polylactic acid, poly(L-lactide) (PLLA),poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(L-lactide-co-D, L-lactide),poly(D,L-lactide-co-trimethylene carbonate), polyglycolic acid,polyhydroxybutyrate (PHB), poly(ε-caprolactone), poly(δ-valerolactone),poly(γ-butyrolactone), poly(caprolactone), polyacrylic acid,polycarboxylic acid, poly(allylamine hydrochloride),poly(diallyldimethylammonium chloride), poly(ethyleneimine),polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone,polyethylene, polymethylmethacrylate, carbon fibers, poly(ethyleneglycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers, poly(ethyleneterephthalate)polyamide, and copolymers and mixtures thereof.Additionally, the PDGF may be enclosed in ceramic materials such astricalcium phosphate, hydroxyapatite, calcium sulphates, or variationsthereof. In addition osmotic pumps may be used to provide protectedrelease of the protein.

The protected PDGF composition is then placed over the tear and issutured in place using the same sutures to do the side-to-side repair ofthe tear, and thereby restore the tissue over the top of the humeralhead. A second protected PDGF composition is then inserted in the samemanner as the first PDGF composition. The second protected PDGFcomposition is placed between the humeral head and the repaired cufftissue. The repaired cuff tissue is then fixed to the site it originallytore away from preferably using suture anchors. The sutures are thensewn through the second protected PDGF composition and torn edge of thecuff to complete the repair.

At the conclusion of the procedure, any incisions are closed usingabsorbable or removable sutures. The patient's shoulder is placed into apostoperative sling to protect the shoulder during the earlypostoperative period.

Absorbable suture anchors or implants are gradually absorbed and thesutures attached are incorporated into the healing tissues. Whenmetallic anchors are used (a matter of surgeon preference), these areburied in the bone, and do not affect the integrity of the bone or theshoulder joint.

Example 5 Treatment of Rotator Cuff Injuries with β-TCP/PDGFCompositions

This study evaluated the efficacy of compositions comprising a rhPDGF-BBsolution combined with a biocompatible matrix comprising β-tricalciumphosphate and type I collagen for the treatment and/or repair of rotatorcuff injuries.

Study Design

Nine (9) adult female sheep were used in the present study. Six of theanimals were administered a test composition comprising a 0.3 mg/mlrhPDGF-BB solution combined with a biocompatible matrix comprisingβ-tricalcium phosphate and type I collagen. As provided herein, 0.3mg/ml rhPDGF-BB solutions was prepared by diluting stock rhPDGF-BBsolutions with 20 mM sodium acetate buffer. The β-TCP was in particulateform, the particles having an average diameter ranging from about 75 μmto about 300 μm. Moreover, the type I collagen was present in an amountof about 20 weight percent of the biocompatible matrix. The remainingthree animals were administered a control composition comprising a 20 mMsolution of sodium acetate buffer combined with a biocompatible matrixcomprising β-tricalcium phosphate and type I collagen.

As part of the study, the animals underwent a period (two weeks) oftendon detachment from the humerus, allowing degenerative changes tobegin in the infraspinatus tendon. The degenerative changes were similarto those observed clinically in rotator cuff injuries. After two weeks,the animals underwent a tendon reattachment procedure in which theinfraspinatus tendon was reattached to the humerus. As provided herein,six of the animals received the test composition at the site of tendonreattachment and the remaining three animals received the controlcomposition at the site of tendon reattachment. All animals were allowedto heal for six weeks. At the six week point, all the animals wereimaged with MRI. Subsequent to imaging, all the animals were humanelysacrificed and biomechnical analysis was performed on three of theanimals receiving the test composition and three of the animalsreceiving the control composition.

Surgical Protocol

All animals were determined to be Q-fever negative prior to being placedin this study. Food was withheld from each animal 24 to 48 hours priorto the procedures and water was removed the morning of surgery. Eachanimal was given a general health evaluation (subject to visualobservation for attitude, activity, and ease in respiration, freedom fordiarrhea and nasal discharge) prior to being placed on the study.Respiratory infection, temperature elevations, observed depression,lameness or anatomical abnormality resulted in rejection of anindividual animal from the surgical procedure. Each animal was weighedwithin 7 days prior to the procedure. Blood was collected for a CBC andChemistry Profile within 7 days of surgery. Twelve animals were examinedand all were found to be acceptable candidates for surgery.

Acepromazine maleate 0.075 mg/kg and Buprenorphine 0.005-0.01 mg/kg wereadministered im prior to anesthetic induction. An intravenous injectionconsisting of Diazepam 0.22 mg/kg and Ketamine 10 mg/kg was given forinduction of general anesthesia. A cuffed endotracheal tube was placedand general anesthesia maintained with Isoflurane 0.5-5% delivered inoxygen through a rebreathing system. Each animal was placed on aventilator to assist respiration. A catheter was placed in a peripheralear vein of each animal. A stomach tube was placed if regurgitationoccurred.

All surgical procedures were conducted utilizing routine aseptictechniques. Pre-operative preparation was conducted in the animalpreparation room adjacent to the operating room. The appropriateshoulder and surrounding areas of each animal were prepared by clippingthe area. Each animal was then moved to the operating room, and the areacleansed with chlorhexidine scrub alternating with 70% isopropyl alcoholthree times and painted with iodine solution. Each animal was thendraped for sterile surgery. Lactated Ringer's Solution (LRS) wasintravenously infused at a rate of about 10-20 ml/kg/hr during surgery.Cefazolin 1-2 gram was intravenously administered prior to the initialincision, and 0.5 g Cefazolin was placed in the flush solution for eachsurgery.

A. Tendon Detachment Surgical Procedure

A 15 cm curved incision was made over the posterolateral aspect of theshoulder joint. The incision was deepened, and the acromial portion ofthe deltoid muscle identified. The muscle was elevated at its cranialedge to expose the tendinous insertion of the infraspinatus muscle andits insertion into the proximal part of the humerus bone. Theinfraspinatus tendon was detached sharply from its insertion on theproximal humerus. The tendon was then wrapped in a 5 cm×3 cm sheet ofPRECLUDE® (W.L. Gore & Associates, Newark, Del.). This allowed diffusionof nutrients to the tendon but inhibits scarring of the tendon to thesurrounding tissues. The incision was closed in standard fashion. A 10cm diameter softball was affixed to the hoof of the limb associated withthe operation to inhibit weight bearing for 7 weeks post-operation.Cefazolin lg was intravenously administered. A 100 μg fentanyl patch wasplaced on each animal.

B. Tendon Reattachment Surgical Procedure

Two weeks following initial surgery, each animal received a secondprocedure to repair the shoulder which underwent tendon detachment. Thesame surgical approach was used as in the first surgery.

In all animals, a moderate degree of tendon retraction was observed aswell as adhesions to the tendon and muscle belly except for the regionprotected by PRECLUDE. These adhesions were dissected, freeing themuscle prior to reattachment in each animal. In preparation forinsertion of bone anchors, the surface of the greater tuberosity wasdecorticated with a bone rongeur so as to create a bleeding bone surfaceand provide access to mesenchymal stem cell migration. At the same time,a 4 mm drill hole, approximately 10 mm deep was created within thedecorticated area to provide a reservoir for test or control material.Two self-tapping suture anchors (15 mm in length by 5 mm wide) were thenscrewed into bone and on either side of the drill hole, and placedapproximately 12 mm apart. Following insertion of the anchors, thetendon was unwrapped and mobilized by blunt dissection. The tendon wasgrasped with no. 2 ETHIBOND® (Johnson and Johnson) braided polyestersuture in a modified Mason-Allen technique.

Prior to the reattachment surgery, stock PDGF-BB solutions (1.0 mg/mL, 5mL total) (Lot # AAI-0022006-5A) were diluted 1:3 in 20 mM acetatebuffer (pH 6.0) to a final concentration of about 0.3 mg/mL. Theresidual amounts of stock diluted PDGF-BB solutions were assayed by UVspectrophotometry to confirm the final solution concentration asprovided in Table 1.

TABLE 1 Stock and diluted solutions of PDGF Final Concentration(Undiluted) (0.53 Extinction PDGF Tube OD Stock Tube Coefficient) FinalPDGF-BB A 0.125 BMIG9 0.235849057 Sample Dilutions B 0.188 BMIG100.354716981 C 0.207 BMIG11 0.390566038 D 0.187 BMIG12 0.352830189 E0.173 BMIG13 0.326415094 F 0.181 BMIG15 0.341509434 G 0 Acetate Buffer 0H 0 Acetate Buffer 0 I 0 Saline** 0 Stock PDGF-BB BMIG9 0.57 N/A1.075471698 Solutions BMIG10 0.515 N/A 0.971698113 BMIG11 0.478 N/A0.901886792 BMIG12 0.5 N/A 0.943396226 BMIG13 0.523 N/A 0.986792453BMIG15 0.508 N/A 0.958490566 * Equation for determining proteinconcentration (OD * dilution factor)/0.53 = Final concentration (mg/mL)**Saline was used to hydrate β-TCP/type I collagen matrix due to ashortage of acetate buffer

Each animal received approximately 1 cc of a β-TCP/type I collagenmatrix hydrated with either a 20 mM sodium acetate buffer solution (pH6.0) or a 0.3 mg/mL rhPDGF-BB solution. For each animal, a fresh stockof acetate buffer or rhPDGF-BB stock solution was opened and diluted toproduce the hydrating solution. Hydrating solutions A-I used in thepreparation of compositions for the treatment of the nine animals in thestudy are provided in Table 1. Moreover, Table 2 provides the assignmentof each of the hydrating solutions (A-I) to the animals in the study.

TABLE 2 Assignment schedule of control and test compositions Applieddose of Test or Animal Hydrating Solution Control Composition G2437 G 1cc G1636 A 1 cc G1637 E 1 cc G1627 F 1 cc G2923 H 1.5 cc   G2054 I 1 ccG2051 D 1 cc G1647 C 1 cc G2922 B 1 cc

Using aseptic technique, a 1 cc β-TCP/type I collagen brick was hydrated(1:3 ratio, β-TCP/type I collagen:PDGF solution) in a sterile dish with3 cc of rhPDGF-BB solution or acetate buffer. In the case of hydratingsolution “I”, an insufficient volume of acetate buffer was available,and sterile saline was substituted for acetate buffer. The material wasmixed with a sterile stainless spatula for approximately 1 minute untila homogenous consistency was achieved. The spatula was then used to loada 3 cc syringe barrel with as much of the test or control material aspossible, the plunger inserted, and the graduated volume noted.

Approximately 1 cc of the test or control composition was applied acrossthe bony tendon footprint and within the drill hole created between theanchors using a 3 cc syringe. Due to muscle and tendon wasting, andretraction, a modest amount of force was required to reapproximate thetendon anteriorly with its footprint. The tendon was then permanentlytied to the anchor resting on bed of bleeding bone and test composition.For all animals, the process of suturing the tendon into place causeddisplacement of approximately 300 uL of the test or control compositioninto the space adjacent to the tendon. The wound was then closed in astandard fashion, and Cefazolin lg iv was administered along with a 100ug fentanyl dermal patch placed on each animal.

C. Post-Operative Care

The animals were returned to the pre/post operating room wherepostoperative monitoring was continued. In this environment, the animalswere monitored during anesthetic recovery for physiological disturbancesincluding cardiovascular/respiratory depression, hypothermia, andexcessive bleeding from the surgical site. Supplemental heat wasprovided as needed. The endotracheal and stomach tubes were removedafter the animals regained the swallow reflex and was breathing on theirown. Cefazolin 1 gram and Buprenorphine 0.005-0.01 mg/kg wereadministered im once postoperatively as the last treatment of the day.Additional analgesic was given as deemed necessary by a staffveterinarian. Long term postoperative monitoring included inspection ofsurgical sites and return to normal physiological function and attitude.Each animal received im injections of antibiotics once daily for 3 days(Naxcel). Each animal was monitored and scored for pain daily for atleast 5 days. Pain evaluation was according to The Assessment of Pain inSheep and Goats after Orthopedic Surgery. Body temperature, pulse, andrespiration were recorded for each animal on days 1-3. General healthassessments were conducted daily for at least 14 days. After that time,animal health was monitored and changes to health status were noted. Thesame pre and post operative procedures were followed for both of thesurgeries. The softball/casts were changed once during the duration ofthe study at four weeks after the first surgery. Seven weeks after theinitial surgery (five weeks after the second), the softball was removedfrom the operative limb and the animals were allowed full movement ofthe leg.

Magnetic Resonance Imaging

Prior to the terminal procedure, all animals were imaged with a GEHealthcare Signa Hdx 1.5T MR imaging system on the operated shoulder. Inboth axial and coronal orientations, the animals were scanned firstusing a T1 weighted protocol, and second a T2 fat-suppressed protocol.Table 3 provides the imaging orientation and protocol for each animal.

TABLE 3 Imaging orientation and protocol Orientation Protocol T1 AxialFSE-XL, 16 FoV, 3 mm slice thickness, 0.5 mm slice gap, 256 × 192 × Z512matrix, 2 Nex, 8.8 TE, 600TR STIR Axial FSE-XL, 16 FoV, 3 mm slicethickness, 0.5 mm slice gap, 256 × 192 × Z512 matrix, 3 Nex, 60 TE, 4500TR, 150 IR T1 Coronal FSE-XL, 16 FoV, 3 mm slice thickness, 0.5 mm slicegap, 256 × 192 × Z512 matrix, 2 Nex, 8.8 TE, 600TR STIR FSE-XL, 16 FoV,3 mm slice thickness, 0.5 mm slice gap, Coronal 256 × 192 × Z512 matrix,3 Nex, 60 TE, 4500 TR, 150 IR

As determined by independent analysis by two certified radiologistsblinded to the treatment groups, animals treated with the testcomposition comprising a rhPDGF-BB solution combined with a β-TCP/type Icollagen matrix demonstrated superior healing of the infraspinatustendon in comparison to animals treated with the control composition.

Subsequent to imaging, all the animals were humanely euthanized by bolusinjection of pentobarbital (Euthansol B) 100-200 mg/kg. Necropsy andtissue collection were conducted on each euthanized animal forbiomechanical and histological analysis.

Biomechanical Testing

Following sacrifice, the treated and contralateral shoulders of allanimals were dissected for biomechanical testing. All dissectedshoulders were first wrapped in saline soaked gauze, placed inindividual, uniquely identified plastic bags, and frozen to −80° C.until time for testing. During testing, the contralateral shoulders wereused to normalize animal to animal variability. Testing was performedusing a biomechanical testing apparatus model number 150 kN from Instronof Norwood, Mass., in which the free tendon was affixed with acryo-clamp. The humeral head was affixed by means of an intramedullarybolt passed through the humeral head in a clevis device arrangement. Thetendon and humerus were then distracted at a rate of 4 mm/second untilcomplete separation of the tendon and humerus was achieved. The forcewas recorded at 0.02 second increments. Mode of failure was alsorecorded. Table 4 summarizes the results of the biomechanical testingfor each animal in the study.

TABLE 4 Summary of biomechanical testing results Mean Force to SpecimenTreatment File Name Max Load (N) Mode of Failure Failure (N)Contralateral Control G2051R Pull01d 1313 Avulsion 1269 ContralateralControl G1627R Pull02 1108 Avulsion Contralateral Control G2054r Pull05a694 Tendon Tear Contralateral Control G1636L Pull08 1503 Bone FractureContralateral Control G2437L Pull09 1069 Avulsion Contralateral ControlG2923R Pull12 1929 Bone Fracture Matrix G2054L Pull04 367 Tendon Tear543 Matrix G2923L Pull10 625 Tendon Tear Matrix G2437R Pull11 636 TendonTear PDGF G1627L Pull03 1179 Avulsion 994 PDGF G1636R Pull06 960Avulsion PDGF G2051L Pull07 845 Avulsion

By applying a t-test to the above data to compare control compositionversus test composition treated shoulders, a statistically significant(p=0.028) increase in load to failure was observed among animals treatedwith test composition comprising rhPDGF-BB. Table 5 provides a summaryof the statistical analysis.

TABLE 5 Summary of statistical analysis Normality Test: Passed (P =0.648) Equal Variance Test: Passed (P = 0.837) Group Name N Missing MeanStd. Dev. SEM Matrix 3 0 550.000 152.069 87.797 PDGF 3 0 994.000 169.23797.709 Difference: −444.000 T = −3.380 with 4 degrees of freedom. (P =0.028)

The increased load to failure in shoulders treated with the testcomposition indicated that the test composition comprising PDGF provideda stronger tendon reattachment to the bone in comparison to the controlcomposition.

Additionally, all tendons treated with the test composition exhibitedfailure at the tendon insertion by avulsion on the bone, whereas tendonstreated with the control compositions failed in the midsubstance of thetendon through tearing and delamination. This difference in mode offailure suggests that the application of rhPDGF-BB increases the tensilestrength and maturity of the tendon, which does not occur in the controlgroup, resulting in avulsion from the insertion site.

Example 6 Treatment of Rotator Cuff Injuries with β-TricalciumPhosphate/PDGF Compositions

This study evaluated the efficacy of a composition comprising arhPDGF-BB solution combined with a biocompatible type I bovine collagenmatrix for the treatment and/or repair of rotator cuff injuries.

Experimental Design

Sheep were selected as an appropriate animal model for the presentstudy. The biomechanical forces measured in the rotator cuff of sheepapproximate those which occur in the human shoulder. The animals andprotocol used in this study are the current benchmark standard forevaluating rotator cuff repair.

A total of forty (40) animals were studied. All the animals were femaleand skeletally mature as determined by plain film radiography to ensureclosure of the physis. The 40 animals were divided into 5 treatmentgroups as provided in Table 6 below. All animals were randomly assignedto the treatment groups.

TABLE 6 Summary of animal treatment groups Animals Treatment Group (n)rhPDGF-BB Imaging Endpoint 1 Suture 8 0 MRI Biomechanics 2 Matrix +Buffer 8 0 MRI Biomechanics 3 Matrix + Dose I 8 0.3 mg/mL MRIBiomechanics 4 Matrix + Dose II 8 1.0 mg/mL MRI Biomechanics 5 Matrix +Dose III 8 3.0 mg/mL MRI Biomechanics

Animals of all groups underwent two procedures. The first procedure wasa resection of the infraspinatus muscle and cutting of the rotator cufftendon. The second procedure occurred two weeks from the tendondetachment surgery to repair the tendon to bone at its insertion on thehumerus. Reattachment of the rotator cuff tendon was administered withbone anchors as provided herein.

Group 1 received only bone anchors and suture for the reattachment ofthe tendon. In addition to bone anchors and suture, Group 2 received atype I collagen matrix hydrated with sodium acetate buffer (20 mM NaAcetate, pH 6.0), the hydrated collagen matrix positioned at the site oftendon reattachment. Moreover, in addition to bone anchors and suture,Groups 3, 4, and 5 received a type I collagen matrix hydrated with arhPDGF-BB solution (0.3 mg/mL, 1.0 mg/mL, and 3.0 mg/mL, respectively),the hydrated collagen matrix positioned at the site of tendonreattachment. Collagen matrices hydrated and applied to animals inGroups 2-5 were obtained and are commercially available from Kensey NashCorporation of Exton, Pa. under the tradename BIOBLANKET®.

Surgical Protocol

All animals were determined to be Q-fever negative prior to being placedin this study. Food was withheld from each animal 24 to 48 hours priorto the procedures and water was removed the morning of surgery. Eachanimal was given a general health evaluation (subject to visualobservation for attitude, activity, and ease in respiration, freedom fordiarrhea and nasal discharge) prior to being placed on the study.Respiratory infection, temperature elevations, observed depression,lameness or anatomical abnormality resulted in rejection of anindividual animal from the surgical procedure. Each animal was weighedwithin 7 days prior to the procedure. Blood was collected for a CBC andChemistry Profile within 7 days of surgery.

Acepromazine maleate 0.05 mg/kg and Buprenorphine 0.005-0.01 mg/kg wereadministered im prior to anesthetic induction. An intravenous injectionconsisting of Diazepam 0.22 mg/kg and Ketamine 10 mg/kg was given forinduction of general anesthesia. A cuffed endotracheal tube was placedand general anesthesia maintained with Isoflurane 0.5-5% delivered inoxygen through a rebreathing system. Each animal was placed on aventilator to assist respiration. A catheter was placed in a peripheralear vein of each animal. A stomach tube was placed if regurgitationoccurred.

All surgical procedures were conducted utilizing routine aseptictechniques. Pre-operative preparation was conducted in the animalpreparation room adjacent to the operating room. The appropriateshoulder and surrounding areas of each animal were prepared by clippingthe area. Each animal was then moved to the operating room, and the areacleansed with chlorhexidine scrub alternating with 70% isopropyl alcoholthree times and painted with iodine solution. Each animal was thendraped for sterile surgery. Lactated Ringer's Solution (LRS) wasintravenously infused at a rate of approximately 10-20 ml/kg/hr duringsurgery. Cefazolin 1-2 g was intravenously administered prior to theinitial incision and 0.5 g Cefazolin was placed in the flush solutionfor every surgery.

A. Tendon Detachment Surgical Procedure

A 15 cm curved incision was made over the posterolateral aspect of theshoulder joint. The incision was deepened, and the acromial portion ofthe deltoid muscle was identified. The muscle was elevated at itscranial edge to expose the tendinous insertion of the infraspinatusmuscle and its insertion into the proximal part of the humerus bone. Theinfraspinatus tendon was detached sharply from its insertion on theproximal humerus. The tendon was then wrapped in a sheet of PRECLUDE®.This allowed diffusion of nutrients to the tendon but inhibited scarringof the tendon to the surrounding tissues. The incision was closed instandard fashion. A 10 cm diameter softball was affixed to the hoof ofthe limb associated with the operation to inhibit weight bearing for 7weeks post-operation. Cefazolin lg was administered iv. A 100 μgfentanyl patch was placed on the animal.

B. Tendon Reattachment Surgical Procedure

Two weeks following the tendon detachment surgery the animals wereshaved and prepped for surgery. General anesthesia was administered toeach animal as provided hereinabove. The shoulder was approached asdescribed previously. The surface of the tuberosity was roughened with arongeur to create a bleeding bone surface prior to anchor insertion. Twometal suture anchors were used, typically 6 mm in length by 2-3 mm wide(commercially available from Smith and Nephew Endoscopy of Andover,Mass.). Each anchor was screwed in within the boundary of the footprintuntil flush with the humeral surface. The center of both anchors wereplaced approximately 1 cm apart. The tendon was unwrapped and mobilizedby blunt dissection. For animals receiving a type I collagen patchhydrated with acetate buffer or a rhPDGF-BB solution, a No. 2 ETHIBOND®braided polyester suture (Johnson and Johnson) looped through theanchors was first passed through the collagen patch, passed through thetendon, tied with a modified Mason-Allen knot, and pulled over thetendon footprint. The tendon was permanently tied to the anchor and thewound is closed in a standard fashion. FIG. 4 illustrates positioning ofthe collagen patch at the site of tendon reattachment.

Prior to implantation and to facilitate handling and placement, eachcollagen patch was cut in half using a sterile ruler and scalpel tocreate two (2) 1 cm² collagen patches. Subsequent to cutting and priorto implantation, each 1 cm² collagen patch was hydrated by applicationof 150 μL of acetate buffer or rhPDGF-BB solution for 5 minutes untilcompletely saturated. As provided herein, all animals that received thetype I collagen patch have sutures from each anchor passed through each1 cm² patch with a needle driver, and the patch pushed along the suturesuntil it is positioned over the decorticated footprint.

The tissue layers of each animal were subsequently closed, the edges ofthe skin incision re-apposed, sutured, and stapled. Each animal receiveda post-operative analgesic to minimize pain. The operated limb had asoftball placed under the hoof during this post-operative period, whichallowed limited movement for five weeks. Five (5) weeks following thereattachment procedure, all animals were imaged by MRI, and the scansare assessed by a radiologist. At the time of MRI, the softballs andcasts were removed. The following week, six (6) weeks post-reattachment,all animals were humanely sacrificed, and the rotator cuff and attachedinfraspinatus tendon collected for biomechanical testing.

In Vivo Observations and Measurements Clinical Observations

Animals were observed daily until the terminal procedure. During thefirst 14 days post-operatively, the animals were observed for generalattitude, appetite, urine/fecal production, appearance of the surgicalsite and respirator stress. Temperature, pulse and heart rate wererecorded for the first 3 days post-operatively. Pain was assessed for aminimum of 7 days post-operatively according to the Evaluation Form forthe Assessment of Pain in Sheep and Goats. Antibiotics were administeredto an animal if infection developed at the surgical site and was notedin the observations. Body weights were recorded prior to each surgicalprocedure and before the terminal procedure. Food consumption wasqualitative. Animals were monitored daily and the degree of appetite isrecorded.

MRI Imaging

MRI scans were taken of each animal at the direction of the studysponsor. Each animal was sedated and then placed in a lateral decubitusposition with the operated shoulder down. Each animal was restrained tothe table, monitored, and scanned for approximately 20 minutes. Table 7provides MRI sequence protocols. After scanning, each animal wasrevived. Scans are forwarded to designated radiologists for a blindreview.

TABLE 7 MRI sequence protocols Sagittal PD Sagittal T1 tr 1000 tr 500 te10 te 10 etl 4 etl 2 rbw 31 rbw 25 fov 14 fov 14 slice thick 4 slicethick 4 slice gap 0 slice gap 0 mtrx 512 × 512 mtrx 512 × 512 nex 3 nex2 pulse seq fse pulse seq fse Coronal PD Fat/Sat Sagittal PD Fat/Sat tr1450 tr 1350 te 11 te 11 etl 4 etl 4 rbw 31 rbw 31 fov 14 fov 14 Xslicethick 4 slice thick 4 slice gap 0 slice gap 0 mtrx 512 × 512 mtrx 512 ×512 nex 3 nex 3 pulse seq fse pulse seq fse

Necropsy

Eight (8) weeks following the tendon detachment surgery all animals werehumanely sacrificed for tissue collection. The humerus and approximatelyfour (4) inches of the humeral shaft were collected along with theattached infraspinatus tendon and approximately two (2) inches of muscledistal to the myotendinous junction. All tissues were promptly wrappedin saline soaked gauze, double wrapped in labeled sealed plastic bags,and frozen to −20° C. until they were thawed for biomechanical testing.

Biomechanical Testing

Biomechanical testing was performed by the Rhode Island HospitalOrthopedic Foundation, Inc. During biomechanical testing, contralateralshoulders of the animals were used to normalize animal to animalvariability. Testing was performed using an biomechanical testingapparatus model number 150 kN from Instron of Norwood, Mass., in whichthe free tendon was affixed with a cryo-clamp. The humeral head wasaffixed by means of an intramedullary bolt passed through the humeralhead in a clevis device arrangement. The tendon and humerus were thendistracted at a rate of 4 mm/second until complete separation of thetendon and humerus was achieved. The force was recorded at 0.02 secondincrements. Mode of failure was also recorded.

Shoulders treated with a type I collagen patch saturated with arhPDGF-BB solution demonstrated an improvement in the ultimate force totendon separation from the shoulder. Table 8 summarizes the forcerequired to separate the reattached tendon from the shoulder as apercentage of the force required to separate the normal contralateralfrom its insertion into the humerus.

TABLE 8 Summary of Biomechanical Testing Group % of Normal 1 (Sutureonly) 59.6 3 (Matrix, 0.3 mg/ml PDGF) 79.8 4 (Matrix, 1.0 mg/ml PDGF)75.3 5 (Matrix, 3.0 mg/ml PDGF) 73.5As displayed in Table 8, force to tendon separation was higher forshoulders treated with a type I collagen patch saturated with arhPDGF-BB solution indicating a stronger reattachment of the tendon tothe bone in comparison with suture only.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

1-18. (canceled)
 19. A method for treating damaged or injured tissuecomprising: providing a composition consisting essentially of ascaffolding material having a solution of platelet-derived growth factor(PDGF) disposed therein, the scaffolding material consisting essentiallyof i) collagen or ii) collagen and a biocompatible binder, and thesolution having a PDGF concentration ranging from about 0.05 to about5.0 mg/mL, and applying the composition to the damaged or injuredtissue.
 20. The method of claim 19, further comprising debridingnon-viable tissue prior to applying the composition to the damaged orinjured tissue.
 21. The method of claim 19, wherein the total amount ofPDGF applied to the tissue during a treatment period ranges from about 1μg to about 50 mg.
 22. The method of claim 19, wherein the total amountof PDGF applied to the tissue during a treatment period ranges fromabout 10 μg to about 25 mg.
 23. The method of claim 19, wherein thetotal amount of PDGF applied to the tissue during a treatment periodranges from about 100 μg to about 10 mg
 24. The method of claim 19,wherein the step of providing the composition comprises disposing thesolution of PDGF in the scaffolding material.
 25. The method of claim19, wherein the scaffolding material provides a framework for new tissuegrowth to occur.
 26. The method of claim 19, wherein the collagen is acollagen patch, pad, gel or paste.
 27. The method of claim 19, whereinthe collagen is a collagen patch or pad.
 28. The composition of claim19, wherein the collagen is a type I, type II or type III bovinecollagen.
 29. The method of claim 19, wherein the collagen is solubletype I bovine collagen.
 30. The method of claim 19, wherein the collagenhas a density ranging from about 0.75 g/cm3 to about 1.5 g/cm3.
 31. Themethod of claim 19, wherein the collagen has a wet tear strength rangingfrom about 0.75 pounds to about 5 pounds
 32. The method of claim 19,wherein the collagen is capable of absorbing water in an amount rangingfrom about 1× to 15× the mass of the collagen.
 33. The method of claim19, wherein the scaffolding material is porous.
 34. The method of claim19, wherein the solution has a PDGF concentration ranging from about 0.1to about 1.0 mg/mL.
 35. The method of claim 19, wherein the solution hasa PDGF concentration ranging from about 0.2 to about 0.4 mg/mL.
 36. Themethod of claim 19, wherein the solution has a PDGF concentration ofabout 0.3 mg/mL.
 37. The method of claim 19, wherein the scaffoldingmaterial comprises pores having a size distribution between about 1microns to about 1,000 microns.
 38. The method of claim 19, wherein thePDGF solution is disposed within the pores of the scaffolding material.39. The method of claim 19, wherein the biocompatible binder is selectedfrom the group consisting of alginic acid, arabic gum, guar gum, xanthamgum, gelatin, chitin, chitosan, chitosan acetate, chitosan lactate,chondroitin sulfate, N,O-carboxymethyl chitosan, α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, sodium dextran sulfate, fibrin glue,lecithin, phosphatidylcholine derivatives, glycerol, hyaluronic acid,sodium hyaluronate, methylcellulose, carboxymethylcellulose,hydroxypropyl methylcellulose, hydroxyethyl cellulose, a glucosamine, aproteoglycan, a starch, lactic acid, a pluronic acid, sodiumglycerophosphate, glycogen, a keratin, silk, and derivatives andmixtures thereof.
 40. The method of claim 19, wherein the PDGF isPDGF-BB.
 41. The method of claim 19, wherein the PDGF is recombinanthuman (rh) PDGF-BB.
 42. The method of claim 19, wherein the PDGFsolution comprises a buffer.
 43. The method of claim 42, wherein thebuffer is sodium acetate.
 44. The method of claim 19, wherein the PDGFcomprises a combination of intact rhPDGF-B (1-109) and fragments thereof45. The method of claim 19, wherein the PDGF comprises at least 65% ofintact rhPDGF-B (1-109).
 46. The method of claim 19, wherein the damagedor injured tissue is a soft tissue.
 47. The method of claim 19, whereinthe damaged or injured tissue is a wound.
 48. A method for treatingdamaged or injured tissue comprising: providing consisting of ascaffolding material having a solution of recombinant humanplatelet-derived growth factor-BB (rhPDGF-BB) disposed therein, whereinthe scaffolding material is a porous collagen patch or pad, and thesolution has an rhPDGF-BB concentration ranging from about 0.1 to about1.0 mg/mL in a buffer, and applying the composition to the damaged orinjured tissue.